The formation of skeletal muscle myotubes requires functional membrane receptors activated by extracellular ATP

Skeletal muscle differentiation follows an organized sequence of events including commitment, cell cycle withdrawal, and cell fusion to form multinucleated myotubes. The role of adenosine 5′-triphosphate (ATP)-mediated signaling in differentiation of skeletal muscle myoblasts was evaluated in C₂C₁₂ cells, a myoblast cell line. Cell differentiation was inhibited by P2X receptor blockers or by degradation of endogenous ATP with apyrase. However, pertussis toxin, known to block only a group of P2Y receptors, did not alter the differentiation process. Cells were heterogeneous in their expression of functional P2X receptors, evaluated by the uptake of fluorescent permeability tracers (Lucifer yellow and ethidium bromide), and by immunofluorescence of P2X₇ receptors. Moreover, xestospongin C, a selective and membrane-permeable inhibitor of IP₃ receptors, inhibited both myotube formation and myogenin expression. Based on these results, we suggest that the known increase in intracellular Ca ²⁺ concentration required for differentiation is due at least in part to Ca²⁺ influx through P2X receptors and Ca²⁺ release from intracellular stores. The possible involvement of P2X receptors and other pathways that might set the intracellular Ca²⁺ at the level required for myoblast differentiation as well as the possible involvement of gap junction channels in the intercellular transfer of second messengers involved in coordinating myogenesis is proposed.